The present invention relates to target cell-specific non-viral vectors, to pharmaceutical compositions that comprise such vectors, and to the use of these vectors in gene therapy.
The aim of gene therapy is to insert a foreign gene(s) into the cells of an organism in order either to switch off defective genes, to replace a defective gene with an intact gene, or to enable these cells to form a protein that possesses a prophylactic or therapeutic effect.
Vectors for insertion of genes into eukaryotic cells, based on viruses, are well known in the art. Viruses have developed a differentiated system by means of which they bind specifically to cells by means of coat proteins, and, after being endocytosed via endosomes, are able to penetrate the membrane of these endosomes and reach the interior of the host cells. Viruses have therefore been used as carriers for inserting foreign genes into the cell. This technology, in its different variations, and the viruses that are used for this purpose, have already been described in detail (see the reviews of Hodgson, Bio/Technology, 3: 222 (1995); and Jolly, Cancer Gene Therapy, 1: 51 (1994)).
The principle underlying this technology is that parts of the viral gene are replaced by the desired foreign gene so that a viral vector is produced. As a rule, viral vectors are no longer able to replicate, due to the manipulation. However, all the genes that encode the viral coat proteins and regulate the expression of these viral genes must be present to enable these viral vectors to replicate.
It has been found, however, that viral vectors can give rise to problems, particularly when being used in humans. There is the danger of recombination with wild-type viruses of the same species, as a result of which pathogenic viruses might be produced. Furthermore, viral coat proteins can trigger immune reactions in the recipient. As viral vectors take the same route of infection in the cell as do the corresponding wild viruses, there is the danger of the host genes being mutated as a result of the foreign genes being integrated into the host chromosomes (activation of quiescent genes, destruction of active genes).
A further disadvantage of viral vectors is that the geometry of the viruses restricts their ability to accommodate many foreign genes.
In view of these limitations and dangers of viral vectors, attempts have been made to find virus-independent methods of inserting genes into cells. The principle underlying one of these methods is fusing the negatively charged cell membrane with the negatively charged gene so that the gene is taken up by the cell, and penetrates into the cytoplasm through the endosomal membrane or the lysosomal membrane. Apart from developing physical (enclosure of gene particles, osmotic, thermal or electrical alterations to the cell membrane) or chemical (organic solvents, detergents, enzymes) methods for altering the cell membrane, gene carriers have been developed that mediate fusion of the genes with the cell membrane. These carriers include liposomes, cationic polypeptides, dendrimeric polymers or cationic amphiphilic substances (for reviews, see Behr, Bioconjugate Chem., 5: 382 (1994); Afione et al., Clin. Pharmakokinet., 28: 181 (1995) and Felgner, Adv. Drug Delivery Rev., 5: 163 (1990)).
Synthetic cationic amphiphilic substances, such as dioleoyloxypropyltrimethylammonium bromide (DOTMA) in a mixture with dioleoylphosphatidylethanolamine (DOPE) or lipopolyamine (see Behr above), have gained considerable importance in this type of charged gene transfer. The mechanism of action of these cationic amphiphilic substances or substance mixtures is that, due to an excess of cationic charge, they both complex with the negatively charged genes and bind to the anionic cell surface. The amphiphilic character of these carriers leads to fusion with the cell membrane. However, the transfection rate which can be achieved is still markedly less than when using viral vectors. Furthermore, the excess cationic charge on the complexes composed of non-viral carriers and DNA is neutralized, after in-vivo administration, by anionic biological substances (proteins, heparins, etc.), thereby impairing binding to cells.
It, therefore, is an object of this invention to provide a means for inserting a foreign gene into a eukaryotic cell that avoids the drawbacks of prior art methods. This and related objects have been achieved by the invention described below.